Regulation of Autotrophic CO ₂ Fixation in the Archaeon Thermoproteus neutrophilus

Abstract

Thermoproteus neutrophilus, a hyperthermophilic, chemolithoautotrophic, anaerobic crenarchaeon, uses a novel autotrophic CO 2 fixation pathway, the dicarboxylate/hydroxybutyrate cycle. The regulation of the central carbon metabolism was studied on the level of whole cells, enzyme activity, the proteome, transcription, and gene organization. The organism proved to be a facultative autotroph, which prefers organic acids as carbon sources that can easily feed into the metabolite pools of this cycle. Addition of the preferred carbon sources acetate, pyruvate, succinate, and 4-hydroxybutyrate to cultures resulted in stimulation of the growth rate and a diauxic growth response. The characteristic enzyme activities of the carbon fixation cycle, fumarate hydratase, fumarate reductase, succinyl coenzyme A (CoA) synthetase, and enzymes catalyzing the conversion of succinyl-CoA to crotonyl-CoA, were differentially downregulated in the presence of acetate and, to a lesser extent, in the presence of other organic substrates. This regulation pattern correlated well with the differential expression profile of the proteome as well as with the transcription of the encoding genes. The genes encoding phosphoenolpyruvate (PEP) carboxylase, fumarate reductase, and four enzymes catalyzing the conversion of succinyl-CoA to crotonyl-CoA are clustered. Two putative operons, one comprising succinyl-CoA reductase plus 4-hydroxybutyrate-CoA ligase genes and the other comprising 4-hydroxybutyryl-CoA dehydratase plus fumarate reductase genes, were divergently transcribed into leaderless mRNAs. The promoter regions were characterized and used for isolating DNA binding proteins. Besides an Alba protein, a 18-kDa protein characteristic for autotrophic Thermoproteales that bound specifically to the promoter region was identified. This system may be suitable for molecular analysis of the transcriptional regulation of autotrophy-related genes.

Reclassification of Thermoproteus neutrophilus Stetter and Zillig 1989 as Pyrobaculum neutrophilum comb. nov. based on phylogenetic analysis

“…Several species are facultative chemoautotrophs and most exhibit robust growth under organotrophic conditions in media supplemented with proteinaceous substrates (e.g. tryptone); P. neutrophilum is an exception in this respect, but chemotrophic growth on hydrogen, elemental sulfur, and carbon dioxide can be enhanced with addition of organic acids (Ramos-Vera et al, 2010;Schäfer et al, 1986). The G+C content of genomic DNA, as determined by complete genome sequencing, is 50-60 mol%.…”

Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

“…The Sulfolobales-specific training set included the HHC pathway genes that were significantly upregulated under autotrophic conditions in the transcriptomic analyses of M. sedula (7,14) (see Table S1 in the supplemental material). For the Thermoproteales group, the training set included the DHC pathway genes from the comparative proteome and enzyme activity studies of autotrophically and heterotrophically grown cells of T. neutrophilus (15), including Tneu_0418 to Tneu_0422, Tneu_1797 to Tneu_1795, and Tneu_1204. For each training set of genes, we determined orthologs in related genomes, analyzed the potential operon structure of their loci, and collected their upstream DNA regions.…”

“…Regulon reconstruction for the DHC autotrophic pathway in Thermoproteales was started with a DNA motif search for genes in T. neutrophilus that were previously determined by comparative proteomic analysis and enzyme activity measurement to be autotrophically induced (15). Thus, we collected upstream regions of the ppcA, ssr, scr-4hbl, 4hbd-frdAB, ppsA, sucCD, mdh, hpcD, and fumAB genes in T. neutrophilus and their orthologs in other Thermoproteales genomes (see Table S3 in the supplemental material).…”

“…Genes encoding four key enzymes from the DHC pathway in T. neutrophilus are organized into a single cluster that contains two divergently oriented leaderless transcripts, scr-4hbl and 4hbd-frdA-frdB, and both of these transcripts were significantly downregulated in cells grown in the presence of acetate. A hypothetical protein encoded by the Tneu_0751 gene was found to bind with high affinity to the 127-bp promoter region of the scr-4hbl and 4hbd-frdA-frdB operons (15). Because of the constitutive expression of Tneu_0751, it was proposed that this potential autotrophic regulator responds to some inductor or effector molecule that governs its promoter binding.…”

“…However, the mechanism of transcriptional regulation of the HHC pathway in the Sulfolobales is currently unknown. In T. neutrophilus, the combined analysis of enzyme activity, the proteome, and transcription revealed that most of the DHC enzymes were induced in cultures grown autotrophically on carbon dioxide in comparison with cells grown in the presence of organic substrates, such as acetate (15). Genes encoding four key enzymes from the DHC pathway in T. neutrophilus are organized into a single cluster that contains two divergently oriented leaderless transcripts, scr-4hbl and 4hbd-frdA-frdB, and both of these transcripts were significantly downregulated in cells grown in the presence of acetate.…”